Saliva assay technique for heavy metal

ABSTRACT

A method for determining heavy metal loading in a subject includes collecting a saliva sample from the subject containing a concentration of a heavy metal. The saliva sample is subjected to inductively coupled plasma mass spectrometry to yield a heavy metal loading measurement for the subject. The saliva sample is readily collected on a substrate absorbing a preselected amount of saliva such as filter paper. As the amount of saliva necessary to saturate a given volume of substrate is known, the volume of saliva within a substrate is also known. The resulting heavy metal loading measurement is readily correlated with a blood level for the heavy metal in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/678,769 filed Feb. 26, 2007, which claims priority of U.S.Provisional Patent Application Ser. No. 60/776,626 filed Feb. 24, 2006,the entire content of each of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention in general relates to determining the quantity ofheavy metal within an organism and in particular to a noninvasive salivaassay for heavy metal.

BACKGROUND OF THE INVENTION

Heavy metal poisoning remains a public health concern despite efforts toeliminate heavy metal usage. While efforts to remove tetraethyl leadfrom gasoline, paint and other consumer products have helped reduce bodyloading of lead, environmental exposure to lead persists in paint chips,dust and lead-containing kitchenware such as glass, ceramic glazes andmetallic foils.

Mercury represents another heavy metal that tends to bioaccumulate.Mercury exposure has been traced to contaminants found within coal andare noted to bioaccumulate, especially in fish.

The symptoms of heavy metal poisoning are well known, includingespecially deleterious effects suffered by children. Upon identificationof high heavy metal loadings within an individual, lifestyle changes andthe administration of chelation agents serve to minimize the deleteriouseffects. However, these treatments can only be put in place aftertesting of an individual for heavy metal.

While various public health organizations and agencies have beensuccessful in performing universal and routine testing of children forheavy metal poisoning, large numbers of individuals are never tested ortested at a frequency that is less than ideal. Compliant testing forheavy metals has met with limited success owing in part to the necessityfor a blood draw requiring skilled personnel, parental consent, blooddraw trauma to the child, and sophisticated infrastructure to processsamples. Saliva has previously been studied as an attractive alternativebodily fluid for the measurement of heavy metal concentrations. However,attempts to correlate salivary lead with that found in blood, bonemarrow or fatty tissue have proven inconsistent.

Thus, there exists a need for a saliva-based assay for heavy metals inorder to increase screening, especially of children. Additionally, thereexists a need for a reproducible saliva sample collection methodology tofacilitate correlation between blood and saliva heavy metalconcentrations.

SUMMARY OF THE INVENTION

A method for determining heavy metal loading in a subject includescollecting a saliva sample from the subject containing a concentrationof a heavy metal. The saliva sample is subjected to inductively coupledplasma mass spectrometry to yield a heavy metal loading measurement forthe subject. The saliva sample is readily collected on a substrateabsorbing a preselected amount of saliva such as filter paper. As theamount of saliva necessary to saturate a given volume of substrate isknown, the volume of saliva within a substrate is also known. Theresulting heavy metal loading measurement is readily correlated with ablood level for the heavy metal in the subject.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is detailed with respect to the following figureswhich show representative and nonlimiting attributes of the presentinvention:

FIG. 1 is a plot of lead levels as measured in micrograms per deciliteraccording to the present invention for 5 double-blind samples asdetermined by inductively coupled mass spectrometry;

FIG. 2 is a plot of lead level in micrograms per deciliter for 21double-blind samples after correction for the dilution of lead in salivarelative to blood;

FIG. 3A is a plot of lead level in micrograms per deciliter correctedfor saliva dilution for baseline tolerable levels of lead along with 2samples, 5 and 11, indicating lead levels that have reached a dangerouslevel;

FIG. 3B is a plot of lead level in micrograms per deciliter fordouble-blind sample sets on 25 samples, one each of saliva and blood;and

FIG. 4 is a plot of lead level in micrograms per deciliter normalized tothat expected to be found in blood based on saliva sampling for 39double-blind saliva tests.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention has utility as a noninvasive method of determiningheavy metal levels within an individual subject. The present inventionprovides information about heavy metal within an individual subject fromsaliva in contrast to prior art techniques that have relied on invasivesample collection techniques associated with collection of blood ortissue biopsy. The collection of saliva affords considerable advantagesin requiring less skill and precaution in the collection of the sampleas compared to blood or biopsy samples. The present invention inestablishing a reliable correlation between blood and salivaconcentration of a heavy metal has overcome the inconsistenciesassociated with prior art saliva detection methodologies.

As used herein a “subject” is defined to include a mammalian or aviancreature specifically including a human, cow, horse, sheep, dog, cat,horse and chicken. While the collection of a saliva sample as detailedhereafter is in regard to a human subject, it is appreciated that othersaliva samples are readily obtained from nonhuman subjects.

Saliva collection is performed according to the present invention in anynumber of conventional procedures. Saliva is readily collected in avial, with an absorbent swab, or pipetted from the buccal cavity onto anabsorbent substrate. The simplicity of collecting a saliva sample allowsfor an untrained individual to collect such a sample. Preferably, thesample is brought into contact with a preservative so as to maintain thesaliva sample integrity during transport to a measurement facility.Saliva sample collection techniques and preservatives operative hereinare detailed in U.S. Pat. No. 5,968,746.

In an alternative embodiment, a kit is provided for saliva collectionthat includes a pipette and a piece of filter paper. The pipette isinserted into an individual's mouth and a sample of saliva collected.Optionally, a salivation stimulating substance coats the pipette tip.The saliva stimulating substance illustratively includes ascorbic acid,citric acid, and other known stimulants. With the collection of at leastapproximately 0.3 milliliters of saliva within the pipette, the salivais expelled onto the filter paper. Optionally, the filter paper istreated with a substance that delineates the boundaries of a droplet ofsaliva thereon. A boundary delineating substance illustratively includesstarch, a pH indicator, and a vegetable dye. The filter paper has aknown volume saliva absorption per unit area and as such, a measurementof a heavy metal from a preselected unit area of filter paper correlateswith concentrations in saliva of the investigated method. Saliva driedonto filter paper is readily eluted and measured for the amount of heavymetal found therein.

Regardless of the method of saliva collection, the sample is placed in abioseal package and assigned a chain of custody number corresponding tothe sample. Preferably, the sample is collected in the form of anabsorptive paper strip with a predetermined quantity of test strip paperbeing removed from the test strip and placed into a sample preparationbottle. The predetermined amount of saliva saturated test strip isplaced in a quantity of medical grade nitric acid within the samplepreparation bottle with the test strip allowed to digest for apredetermined amount of time. By way of example, a 1 centimeter squarearea of saliva saturated test strip immersed in 5 milliliters of 20%nitric acid is adequately digested after approximately 20 minutes. Thesample preparation bottle contents are then removed, optionally filteredand inserted into vials corresponding to predetermined positions withsample coating facilitating correlation of results with the sample.

The sample solution is then drawn into an inductively coupled plasma(ICP) mass spectrometer (MS) (ELAN DRCe ICP mass spectrometer,PerkinElmer). The ICP MS uses the inductively coupled plasma tosuperheat elements passing through the sample analysis chamber todestroy any molecular bonds and ionize the residual atoms. This liquidsample is formed as an aerosol through application of a vacuum prior toencountering the plasma in preference to air entertainment to form anaerosol so as to enhance signal-to-noise ratio. The ions formed throughexposure to the plasma travel through a quadruple mass spec modulated tocollect isotope ions associated with the heavy metal of interest. By wayof example, lead isotopes 206, 207, 208 masses have a natural abundanceof approximately 24:22:52 atomic percent in addition to providing aquantitative measure of lead within the sample also provides an identitysignature when isotope percentages correspond with a particular exposuresuch as that found in specific industrial working arenas. A similarcorrelation can be made for mercury isotopes having masses 198, 199,200, 201, 202 and 204 that have atomic percent natural abundance ofabout 10:17:23:13:30:7. According to the present invention, heavy metalions are readily detected as low as single parts per trillion withstatistically significant values being obtained within the order of 10to 30 parts per trillion. As heavy metal concentrations in blood aretypically measured in micrograms per deciliter, ICP MS is about threeorders of magnitude greater in sensitivity than the 10 parts per millionof lead that corresponds to a microgram per deciliter.

Heavy metals detectable according to the present invention areappreciated to be readily identified within a single scan merely bymodulating the mass spectrometer to collect particular mass species.Heavy metals readily detectable according to the present inventionillustratively include vanadium, chromium, manganese, cobalt, nickel,copper, arsenic, technetium, ruthenium, rhenium, cadmium, tin, antimony,lanthanide series, osmium, mercury, thallium, and combinations thereof.

Intermediate between spectral collection, the system is purged withsolvent and periodic calibration standards.

Correlative analysis between the amount of a heavy metal present in ablood sample and saliva sample from the same subject is possible becauseblood and saliva are made from the same extracellular fluid. The fluidmatrix that carries eosinophils, neutrophils and nutrients via thecirculatory system also carries enzymatic proteins within the digestivetract. Saliva differs from blood in having a higher water content andshorter circulatory life. As a result, saliva contains less of a heavymetal than the corresponding volume blood sample and additionally salivarepresents a better indicator of recent exposure to high levels of aheavy metal associated with residual lead from sources such as diet,environment or inhalation. Upon ingestion of a heavy metal, it isdiluted to the bloodstream to a volume of approximately 7 liters for anadult human subject or other extracellular fluid such as saliva to avolume of approximately 10 milliliters. Heavy metal is often known toleach into the tissues of the mouth and throat to afford a measurementindicating a toxic heavy metal content for the subject that is not asreadily found by blood heavy metal analysis. As a result, saliva testingallows chelation treatment to be performed before heavy metal has fullyentered the bloodstream and been carried to bone marrow or adiposetissue from which removal is problematic.

The present invention is further detailed with respect to the followingnonlimiting examples.

Example 1 Stock Solutions

HPLC grade water is purchased from Pharmacy and used for up to 12 monthsafter receipt. Each bottle of HPLC grade water when opened is used foronly up to 30 days. A 20% nitric acid solution is created by halffilling a 1 liter volumetric flask with HPLC grade water and adding 287ml of 69.7-70% assay trace metals grade nitric acid to the flask andfilling to a volume of 1 L. Mass spectrometer wash solvent is providedusing HPLC grade water to flush lines.

Clinical standard lead is purchased from VWR International at 1000 ppm,100 ug/dl. Linearity standards are prepared at 2, 10, 50, 100, and 500parts per million (ppm) with volumetric glassware and HPLC grade water.A PerkinElmer ELAN DRCe inductively coupled plasma mass spectrometer isenergized and allowed to stabilize for from 60 to 90 minutes.Thereafter, the instrument is prompted to load the lead detectionprotocol setting appropriate parameters for the lead isotopes 206, 207,and 208. Sample times and rinse times for samples set to a preselectedlevel and calibration standards are run using lead calibration linearitystandards at 2, 5, 10, 50, 100, and 500 ppm to establish a correlationcoefficient of greater than 0.999. A test sample is then run to verifyintegration parameters. A reservoir is then filled with HPLC grade waterand a sample tray of a PerkinElmer 192 Auto Sampler is loaded andprogrammed to run settings and tray positions for the Auto Sampler thatcorrespond to sample and standard positions. The Auto Sampler is thenallowed to provide samples and standards to the ICP mass spectrometer.

This process has been extended and validated for the analysis of a rangeof heavy metals detailed herein in blood or saliva. The method iscalibrated in a range from 0.2 ug/dl to about 50 ug/dl. Samples withhigher concentration than the linear range samples should be diluted andrerun as extrapolated data results are considered to be suspect.

Results are calculated from the calibration curve after verifying thatall the controls are in the acceptable range and the correlationcoefficient of greater than 0.999 is obtained. Saliva values arecorrected for diluted factor based on the amount of saliva that can beabsorbed into a 1 cm² piece of filter paper. Positive samples areflagged for resampling and notification of health authorities.

To assure peak response for each standard is within ±25% of the expectedvalue. Each peak response is then calculated and entered into anappropriate peak log with all peak logs plotted on a monthly basis alongwith acceptable high and low ranges. High and low controls arecalculated to heavy metal levels within 5% of the expected values. Allcontrol results are entered into control result logs and plotted on atrend plot on a routine basis such as weekly. All controls that falloutside of the expected range, the entire sample set are rerun aftercorrective actions have been taken with concert notation as tocorrective actions being documented and the motivations therefor.Controls and standards are run at the beginning and end of each sampleset with one control and one blank being run after each 10 samples.

In order to establish a correlation between inventive saliva testing andconventional whole blood lead level testing, a series of 14 blindsamples were collected of whole blood and corresponding saliva samples.FIG. 1 depicts saliva measured corresponding blood lead level inmicrograms per deciliter for the 14 samples as well as the lower valueof the blood lead level determined by conventional techniques. The rangeof blood low and high levels is also provided in FIG. 1.

Example 2 Correspondence of Blood and Saliva Lead Levels Derived fromInductively Coupled Plasma Mass Spectrometry

Twenty-one double-blind sample sets were collected from volunteers. Eachsample set included a whole blood sample and a saliva sample. Applyingthe procedure of Example 1, and accounting for dilution associated withsaliva absorption into filter paper used to collect the saliva sample, ablood lead level is plotted directly in FIG. 2 for the 21 blind sampleswhile the blood level derived by correcting for saliva dilutionassociated with saliva absorbable into a square centimeter of filterpaper is provided in FIG. 2.

Example 3 Correlation of Blood and Saliva Results Including DangerousLevels of Lead

The sampling procedure of Example 1 is repeated with 17 and 25 dual setsof double-blind saliva and whole blood samples. The results measuredlead levels corresponding to blood levels are plotted in FIGS. 3A and3B, respectively, for the 17 and 25 double-blind sample sets withcorrection being made for the amount of saliva being absorbable into 1cm² of filter paper as the saliva collection medium. Based on the highdegree of correlation between blood levels detected in blood and saliva,blood levels are estimated from saliva for a group of 39 unknownsamples, as shown in FIG. 4.

Example 4 Comparison of Blood and Saliva Analysis by ICP MassSpectrometry

Five sets of double-blind samples each including conventional blood drawand a 1 cm² piece of filter paper saturated with saliva were analyzed byICP mass spectrometry according to the procedure of Example 1 with theresults plotted in FIG. 5 as measured directly without correlation forsaliva dilution and absorption into a quantity of filter paper. Evenwithout correlation, the general agreement is noted between saliva andblood samples.

Patent documents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. These documents and publications are incorporatedherein by reference to the same extent as if each individual document orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

The invention claimed is:
 1. A method for determining heavy metalloading in a subject comprising: collecting a saliva sample containing aconcentration of a heavy metal from a subject; acid digesting saidsaliva sample; subjecting said saliva sample to inductively coupledplasma mass spectrometry to yield a heavy metal loading measurement;said heavy metal loading measurement correlating to a blood level ofsaid heavy metal from a blood sample from said subject.
 2. The method ofclaim 1 further comprising determining the level of said heavy metal insaid saliva by comparing said heavy metal loading measurement to astandard curve, said standard curve having a correlation coefficient ofgreater than 0.999.
 3. The method of claim 1 further comprising dilutingsaid sample so that said heavy metal loading measurement is from 0.2μg/dl to 50 μg/dl.
 4. The method of claim 1 wherein said step ofcollecting a saliva sample is by collecting on an absorbent swab or afilter paper.
 5. The method of claim 1 wherein said heavy metal is lead.6. The method of claim 1 further comprising yielding a second heavymetal loading measurement from said step of subjecting.
 7. The method ofclaim 1 wherein said step of acid digesting is in a solution comprisingaqueous nitric acid.
 8. A method for determining heavy metal loading ina subject comprising: subjecting a saliva sample containing aconcentration of a heavy metal from a subject to inductively coupledplasma mass spectrometry to yield a heavy metal loading measurement forsaid subject, said saliva sample collected on a substrate; said heavymetal loading measurement correlating to a blood level of said heavymetal from a blood sample from said subject.
 9. The method of claim 8wherein said mass spectrometer is a quadrupole mass spectrometer. 10.The method of claim 8 wherein the saliva sample is collected on asubstrate absorbing a preselected amount of saliva.
 11. The method ofclaim 8 wherein the substrate is filter paper.
 12. The method of claim11 wherein said filter paper further comprises an indicator.
 13. Themethod of claim 8 wherein said substrate is an absorbent swab.
 14. Themethod of claim 8 wherein said heavy metal is lead.
 15. The method ofclaim 8 further comprising diluting said saliva sample in apredetermined solution volume prior to said step of subjecting.
 16. Themethod of claim 15 wherein said solution is aqueous nitric acid.
 17. Themethod of claim 8 further comprising acid digesting said saliva samplein said solution volume prior to said step of subjecting.
 18. The methodof claim 8 further comprising yielding a second heavy metal loadingmeasurement from said mass spectrometry.
 19. The method of claim 8further comprising correlating the heavy metal loading measurement witha blood level of said heavy metal in the subject.
 20. The method ofclaim 8 wherein said correlating is by direct correlation of bloodlevels and saliva levels of said heavy metal.